Computer-aided drug screening methods, systems, or devices based on sphingolipids and orf2

Abstract

The application discloses a computer-aided drug screening method, system or device based on sphingolipid and ORF2. It is found for the first time that sphingolipid and ORF2 protein of hepatitis E virus are combined in a host cell Golgi, and the combination site is a conformational C1 pocket region at the interface between S and P2 domains of the ORF2 protein. The combination effect can effectively regulate the assembly and release of the hepatitis E virus. Based on this, the application provides a computer-aided drug screening method, system, device and computer readable storage medium based on sphingolipid and ORF2 for the field. The application provides an efficient, accurate and rapid screening method for the research and development of a new drug for preventing or treating the hepatitis E virus, and has a wide application prospect in the screening of the new drug for preventing or treating the hepatitis E virus.

Description

Technical Field

[0001] This invention belongs to the field of computer-aided drug screening technology, specifically relating to a computer-aided drug screening method, system, or device based on sphingolipids and ORF2. Background Technology

[0002] Hepatitis E virus (HEV) belongs to the Hepatoviridae family, and its genome encodes three proteins: ORF1, ORF2, and ORF3. The ORF2 protein exists in two isoforms: a capsid protein and a secreted protein. The capsid ORF2 protein, after dimerization, polymerization, folding, and assembly, can encapsulate the viral RNA genome, forming non-enveloped virions (nHEVs). These are then packaged and processed into quasi-enveloped virions (eHEVs) and released extracellularly. Host lipids play a crucial role in the packaging of nHEVs into eHEVs. However, due to the diverse expression forms and highly complex structures of host lipids, relevant research is currently very limited. Whether there is an interaction between the ORF2 protein and host lipids during HEV infection and replication remains unclear.

[0003] HEV infection is one of the leading causes of acute viral hepatitis worldwide. Clinical statistics show approximately 20 million new HEV infections and 60,000 HEV-related deaths annually. Currently, there are no officially approved antiviral drugs for HEV treatment. Off-label treatments mainly include ribavirin and pegylated interferon-alpha. However, due to the risk of graft rejection caused by immune responses, pegylated interferon-alpha is not suitable for transplant patients, and ribavirin is strictly prohibited for use in pregnant women due to its teratogenic effects. Furthermore, mutations in the viral polymerase can lead to relapse of viral infection after ribavirin treatment. Therefore, developing safer and more effective drugs for the prevention of HEV infection is a pressing clinical challenge. Computer-aided drug design (Computer...) Aided Drug Design (CADD) uses computer-aided computation and simulation techniques to learn from the prior knowledge contained in massive amounts of drug data and uncover the interaction relationships between target molecules and candidate drugs. This allows for the rapid selection of drug-like active molecules from millions of molecules. This significantly reduces the randomness in screening candidate drugs and improves research and development efficiency. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, the present invention aims to provide a computer-aided drug screening method, system, or device based on sphingolipids and ORF2. This computer-aided drug screening technology is used to screen drugs for the treatment or prevention of hepatitis E virus infection.

[0005] To achieve the above objectives, the present invention provides the following technical method: A first aspect of the present invention provides a computer-aided drug screening method based on the interaction between sphingolipids and the ORF2 protein, the method comprising: Obtain data on sphingolipids and ORF2 proteins; The spatial structure of the sphingolipid-ORF2 protein complex was selected, and the binding site of the sphingolipid-ORF2 protein complex was determined as the binding site of the targeted drug. Candidate drugs targeting the binding site were obtained using a computer-aided drug screening method.

[0006] Furthermore, the method for determining the binding site of the sphingolipid-ORF2 protein complex includes: Predicting the interaction domains between sphingolipids and ORF2 protein using bioinformatics methods; Construct n truncated expression mutants of ORF2, where n is a natural number greater than or equal to 1; Based on the n truncated expression mutants of ORF2, it was found that the S and P2 domains of the ORF2 protein are key regions for interaction with sphingolipids. Bioinformatics methods were used to predict that the spatial conformation formed by the interaction between the conformational C1 pocket region at the interface of the S and P2 domains of the ORF2 protein and sphingolipid is the binding site of the sphingolipid-ORF2 protein complex. Construct n mutants with the C1 pocket region mutation, where n is a natural number greater than or equal to 1; Based on the mutants with the n C1 pocket region mutations, the binding sites of the sphingolipid-ORF2 protein complex include amino acid residues of ORF2 protein T170A, N171A, V470A, V501A, and V503A.

[0007] Optionally, the method for obtaining the binding site of the sphingolipid-ORF2 protein complex is immunoprecipitation.

[0008] Optionally, the bioinformatics method includes molecular docking methods or prediction methods based on sequence and structural information.

[0009] Furthermore, the computer-aided drug screening method includes: Obtain the binding site of the sphingolipid-ORF2 protein complex; Based on the spatial structure of the binding site of the sphingolipid-ORF2 protein complex, compounds targeting the binding site of the sphingolipid-ORF2 protein complex were screened from the compound library. The selected compounds are molecularly docked with ORF2 protein or sphingolipid to calculate the affinity or binding energy of the compounds to target ORF2 protein or sphingolipid, and a score is obtained for each compound. Candidate drugs are obtained by sorting the compounds according to the scores.

[0010] Optionally, the compound includes siRNA, shRNA, dsRNA, microRNA, antisense nucleic acid, antibody, polypeptide, protein analog, peptide analog or inorganic compound.

[0011] Furthermore, the drug screening method also includes: Obtain a system for expressing the ORF2 protein; The effectiveness of the compounds was verified by treating the system expressing the ORF2 protein with the compounds obtained from the screening.

[0012] Optionally, the system for expressing ORF2 protein includes a cell system expressing ORF2 protein, a subcellular system expressing ORF2 protein, a tissue system expressing ORF2 protein, a solution system expressing ORF2 protein, an organ system expressing ORF2 protein, or an animal system expressing ORF2 protein.

[0013] A second aspect of the present invention provides a computer-aided drug screening system based on the interaction between sphingolipids and the ORF2 protein, the system comprising: The data acquisition unit acquires data on sphingolipids and ORF2 proteins. The site determination unit selects the spatial structure of the sphingolipid-ORF2 protein complex and determines the binding site of the sphingolipid-ORF2 protein complex as the binding site of the targeted drug. The drug screening unit uses a computer-aided drug screening method to obtain candidate drugs that target the binding site.

[0014] Furthermore, the method for determining the binding site of the sphingolipid-ORF2 protein complex includes: Predicting the interaction domains between sphingolipids and ORF2 protein using bioinformatics methods; Construct n truncated expression mutants of ORF2, where n is a natural number greater than or equal to 1; Based on the n truncated expression mutants of ORF2, it was found that the S and P2 domains of the ORF2 protein are key regions for interaction with sphingolipids. Bioinformatics methods were used to predict that the spatial conformation formed by the interaction between the conformational C1 pocket region at the interface of the S and P2 domains of the ORF2 protein and sphingolipid is the binding site of the sphingolipid-ORF2 protein complex. Construct n mutants with the C1 pocket region mutation, where n is a natural number greater than or equal to 1; Based on the mutants with the n C1 pocket region mutations, the binding sites of the sphingolipid-ORF2 protein complex include amino acid residues of ORF2 protein T170A, N171A, V470A, V501A, and V503A.

[0015] Optionally, the method for obtaining the binding site of the sphingolipid-ORF2 protein complex is immunoprecipitation.

[0016] Optionally, the bioinformatics method includes molecular docking methods or prediction methods based on sequence and structural information.

[0017] Furthermore, the computer-aided drug screening method includes: Obtain the binding site of the sphingolipid-ORF2 protein complex; Based on the spatial structure of the binding site of the sphingolipid-ORF2 protein complex, compounds targeting the binding site of the sphingolipid-ORF2 protein complex were screened from the compound library. The selected compounds are molecularly docked with ORF2 protein or sphingolipid to calculate the affinity or binding energy of the compounds to target ORF2 protein or sphingolipid, and a score is obtained for each compound. Candidate drugs are obtained by sorting the compounds according to the scores.

[0018] Optionally, the compound includes siRNA, shRNA, dsRNA, microRNA, antisense nucleic acid, antibody, polypeptide, protein analog, peptide analog or inorganic compound.

[0019] Furthermore, the method also includes an effectiveness verification unit: obtaining a system expressing the ORF2 protein; and treating the system expressing the ORF2 protein with the screened compound to verify the effectiveness of the compound.

[0020] Optionally, the system for expressing ORF2 protein includes a cell system expressing ORF2 protein, a subcellular system expressing ORF2 protein, a tissue system expressing ORF2 protein, a solution system expressing ORF2 protein, an organ system expressing ORF2 protein, or an animal system expressing ORF2 protein.

[0021] A third aspect of the present invention provides a computer-aided drug screening device based on the interaction between sphingolipids and the ORF2 protein, the device comprising: The system includes a memory for storing program instructions and a processor for invoking the program instructions, which, when executed, implement the computer-aided drug screening method based on the interaction between sphingolipids and the ORF2 protein as described above.

[0022] A fourth aspect of the present invention provides a computer-readable storage medium storing a computer program that, when executed by a processor, implements the computer-aided drug screening method based on the interaction between sphingolipid and ORF2 protein as described above.

[0023] A fifth aspect of the present invention provides a computer program product comprising a computer program that, when executed by a processor, implements the computer-aided drug screening method based on the interaction between sphingolipid and ORF2 protein as described above.

[0024] A sixth aspect of the present invention provides any of the following products: 1) A sphingolipid-ORF2 protein complex, wherein the sphingolipid-ORF2 protein complex is as described above; 2) A drug obtained according to the computer-aided drug screening method based on the interaction between sphingolipid and ORF2 protein as described above.

[0025] A seventh aspect of the invention provides any of the following applications: 1) Application of a protein complex, said complex being a sphingolipid and ORF2 protein complex as described above, said application including: the application of said complex in regulating hepatitis E virus replication and / or release; 2) The use of a drug, wherein the drug is as described above, and the use includes: the use of the drug in the prevention or treatment of hepatitis E virus.

[0026] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: This invention is the first to discover that sphingolipids bind to the hepatitis E virus (HEV) ORF2 protein in the host cell Golgi apparatus, with the binding site being the conformational C1 pocket region at the interface between the S and P2 domains of the ORF2 protein. This binding effectively regulates the assembly and release of HEV. Based on this, this invention provides a computer-aided drug screening method, system, device, and computer-readable storage medium based on the interaction between sphingolipids and the ORF2 protein. This invention provides an efficient, accurate, and rapid screening method for the development of new drugs for the prevention or treatment of HEV, and has broad application prospects in the screening of new drugs for the prevention or treatment of HEV. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of a computer-aided drug screening method based on the interaction between sphingolipids and the ORF2 protein.

[0028] Figure 2The results show the effects of the sphingolipid inhibitor PPMP on ORF2 protein release. Figure A represents the effect of PPMP treatment on ORF2 protein expression in HuH7 cells after HEV virus particle release and reinfection, as shown by immunofluorescence staining. Figure B represents the effect of PPMP on ORF2 protein expression in HuH7-HEV-p6 cells, as shown by Western blotting. Figure C shows the statistical analysis of the effect of PPMP on ORF2 protein expression in HuH7-HEV-p6 cells using Western blotting. Figure D shows the statistical analysis of the effect of PPMP on serum ORF2 antigen levels in a long-clawed gerbil model.

[0029] Figure 3 This is a graph showing the effects of the sphingolipid inhibitor PPMP on ORF2 protein polymerization and HEV assembly in HuH7-HEV-p6 cells. A represents the statistical analysis of HEV RNA expression in the extracellular gradient component; B represents the representative immunoblot graph of HEV ORF2 protein expression in the extracellular gradient component; C represents the statistical analysis of HEV RNA expression in the intracellular gradient component; D represents the representative immunoblot graph of HEV ORF2 protein expression in the intracellular gradient component; E represents the representative immunoblot graph of the effects of PPMP and PDMP on ORF2 protein polymerization; and F represents the statistical analysis of the effects of PPMP and PDMP on ORF2 protein polymerization.

[0030] Figure 4 The diagram shows the results of the co-localization analysis of sphingosine and ORF2 proteins. A is a schematic diagram of the pac-sph structure; B is a representative diagram validating the specificity of pac-sph and β-actin immunoprecipitation; C is a representative diagram validating the specificity of pac-sph and ORF2 immunoprecipitation; D is a representative diagram validating pac-sph and ORF1, ORF2, or ORF3 immunoprecipitation; E is a representative diagram showing the effect of PPMP treatment on the immunoprecipitation of pac-sph and ORF2; and F is a statistical analysis diagram showing the effect of PPMP treatment on the immunoprecipitation of pac-sph and ORF2.

[0031] Figure 5These are the subcellular co-localization results of sphingosine and ORF2 protein. A is a representative image verifying the effectiveness of pac-sph immunofluorescence staining; B is a representative image of pac-sph and ORF2 immunofluorescence staining; C is a representative image of pac-sph and ORF2 immunofluorescence staining in the Golgi apparatus; D is a representative image of pac-sph and ORF2 immunofluorescence staining in endosomes; E is a representative image of pac-sph and ORF2 immunofluorescence staining in the endoplasmic reticulum; F is a representative image of pac-sph and ORF2 immunofluorescence staining in lysosomes; G is a P... Pearson correlation analysis plots showing the effect of PMP treatment on the colocalization of pac-sph and ORF2; H shows the Pearson correlation analysis results of the effect of PPMP treatment on the localization of pac-sph and ORF2 in the Golgi apparatus; I shows the Pearson correlation analysis plots showing the effect of PPMP treatment on the localization of pac-sph and ORF2 in endosomes; J shows the Pearson correlation analysis plots showing the effect of PPMP treatment on the localization of pac-sph and ORF2 in the endoplasmic reticulum; K shows the Pearson correlation analysis plots showing the effect of PPMP treatment on the localization of pac-sph and ORF2 in lysosomes.

[0032] Figure 6 This is a diagram showing the simulation analysis and validation results of the interaction between sphingolipids and ORF2 proteins mediated by linear motifs. A is a schematic diagram of the sphingosine-binding motif sequence; B is a potential sphingosine-binding motif sequence (green) discovered using the motif probability algorithm; C is a schematic diagram of wild-type, secreted, and capsid ORF2 proteins; and D is a representative diagram of pac-sph co-precipitation with secreted and capsid ORF2 proteins, respectively.

[0033] Figure 7 This diagram presents the simulation analysis and validation results of the binding sites between sphingolipids and ORF2 protein monomers. A shows a schematic diagram of different domains of the ORF2 protein; B shows a schematic diagram of the predicted potential sphingolipid interaction pockets on the ORF2 monomers, with different colored amino acids representing four independent pockets (red - CP1, blue - CP2, pink - CP3, green - CP4); C shows a schematic diagram of the predicted interaction pattern between the CP1 monomer and sphingosine in the ORF2 protein; D shows a schematic diagram of the predicted interaction pattern between the CP2 monomer and sphingosine in the ORF2 protein; E shows a schematic diagram of the predicted interaction pattern between the CP3 monomer and sphingosine in the ORF2 protein; F shows a schematic diagram of the predicted interaction pattern between the CP4 monomer and sphingosine in the ORF2 protein; G shows schematic diagrams of truncated ORF2 proteins (S, P1, P2); H shows a representative immunoblot image of truncated ORF2 protein expression; and I shows a representative immunoprecipitation image of pac-sph and truncated ORF2 protein.

[0034] Figure 8This diagram shows the simulation analysis and verification results of the binding sites between sphingolipids and the ORF2 protein dimer. A represents a schematic diagram of potential sphingolipid interaction sites on the ORF2 dimer. Different colored amino acids represent four independent sites (red - C1, blue - C2, pink - C3, and green - C4). B is a schematic diagram of the three-dimensional structure of the ORF2 dimer; C is a schematic diagram of the predicted interaction mode between the C1 domain of the ORF2 dimer and sphingosine; D is a schematic diagram of the predicted interaction mode between the C2 domain of the ORF2 dimer and sphingosine; E is a schematic diagram of the predicted interaction mode between the C3 domain of the ORF2 dimer and sphingosine; F is a schematic diagram of the predicted interaction mode between the C4 domain of the ORF2 dimer and sphingosine; and G is a schematic diagram of the interaction pocket structure between the ORF2 dimer and sphingosine.

[0035] Figure 9 This is a diagram showing the simulation analysis and verification results of the binding site between sphingosine and the C1 domain of the ORF2 protein. A is a schematic diagram of the predicted three-dimensional structure of the interaction between ORF2 amino acids and sphingosine, with one subunit in purple and the other in pink. B is a representative diagram of the co-precipitation of ORF2 M1-M6 proteins with pac-sph. C is a statistical analysis diagram of the co-precipitation of ORF2 M1-M6 proteins with pac-sph. D is a representative diagram of the co-precipitation of pac-sph with the ORF2 mutant M1+M3+M5. E is a schematic diagram of the effect of M1 on the interaction between ORF2 protein and sphingolipids. F is a schematic diagram of the effect of M3 on the interaction between ORF2 protein and sphingolipids. G is a schematic diagram of the effect of M5 on the interaction between ORF2 protein and sphingolipids.

[0036] Figure 10 This diagram shows the effects of ORF2 protein mutations M1, M3, M5, and M1+M3+M5 on HEV RNA and ORF2 protein expression. A is a schematic diagram of the HEV genome; B is a statistical analysis of the effect of ORF2 protein mutations on intracellular HEV RNA expression; C is a statistical analysis of the effect of ORF2 protein mutations on extracellular HEV RNA expression; D is a representative immunoblot diagram showing the effect of ORF2 protein mutations on intracellular and extracellular ORF2 protein expression levels; E is a statistical analysis of HEV RNA expression in intracellular gradient components; F is a representative immunoblot diagram of ORF2 protein in intracellular gradient components; G is a statistical analysis of HEV RNA expression in extracellular gradient components; and H is a representative immunoblot diagram of ORF2 protein in extracellular gradient components.

[0037] Figure 11The diagram shows the effects of M1+M3+M5 mutant HEV infection on HEV RNA and ORF2 protein in long-clawed gerbils. A is a statistical analysis of HEV RNA expression in long-clawed gerbil fecal samples; B is a statistical analysis of HEV RNA expression in the liver, intestine, and kidney of long-clawed gerbils; C is a schematic diagram of pathological staining of long-clawed gerbil liver tissue; and D is a schematic diagram of immunohistochemical staining of ORF2 protein in long-clawed gerbil liver tissue. Detailed Implementation

[0038] To enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0039] In some of the processes described in the specification, claims, and accompanying drawings of this invention, multiple operations are included in a specific order. However, it should be clearly understood that these operations may not be performed in the order they appear herein, or they may be performed in parallel. The operation numbers, such as S101, S102, etc., are merely used to distinguish different operations and do not represent any execution order. Furthermore, these processes may include more or fewer operations, and these operations may be performed sequentially or in parallel.

[0040] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] Figure 1 This is a schematic flowchart of a computer-aided drug screening method based on the interaction between sphingolipids and ORF2 protein, provided by an embodiment of the present invention. Specifically, the method includes the following steps: S101: Obtain data on sphingolipids and ORF2 proteins; In one embodiment, the sphingolipid is a large class of lipids comprising a conserved sphingosine backbone and a modifiable head sequence. Typically, a linear binding motif defined by the sequence [V / I / T / L]XX[V / I / T / L][V / I / T / L]XX[V / I / T / L][F / W / Y] interacts with proteins. The sphingosine molecular structure can be retrieved and downloaded from PubChem.

[0042] In one embodiment, the ORF2 protein is one of three proteins encoded by the HEV genome, existing in two isoforms: a capsid protein and a secretory protein, and containing S, M, and P domains. Multiple subunits assemble around a central region to form an icosahedral structure. The cryo-electron microscopy structure of HEV ORF2 was retrieved and downloaded from the RCSB PDB website and simplified to a dimer.

[0043] In one embodiment, this invention demonstrates that the sphingolipid inhibitor PPMP inhibits the release of ORF2 protein. First, a HuH7-HEV-p6 cell model was constructed by transfecting a Kernow-C1 p6 plasmid carrying the HEV genome into HuH7 cells. The HuH7-HEV-p6 cells were treated with the sphingolipid inhibitor PPMP, and the virus-containing supernatant was collected and re-infected with HuH7 cells. Immunofluorescence staining was used to detect ORF2 protein expression in the HuH7 cells. The results showed that PPMP treatment significantly inhibited ORF2 protein expression (…). Figure 2 A). The effects of PPMP on HEV replication and release were further analyzed using Western blotting, with mycophenolic acid (MPA), which effectively inhibits HEV replication, used as a positive control. The results showed that PPMP had no significant effect on intracellular HEV ORF2 protein expression levels, while significantly inhibiting extracellular HEV ORF2 protein expression. Figure 2 B Figure 2 C). To further verify the effect of PPMP on ORF2 protein expression, we constructed a HEV-infected long-clawed gerbil model and treated it with PPMP. ELISA results showed that PPMP significantly reduced HEV ORF2 protein expression in the serum of long-clawed gerbils. Figure 2 D). These results indicate that sphingolipid inhibitors can significantly suppress the release of ORF2 protein during HEV infection.

[0044] In one embodiment, this invention demonstrates that the sphingolipid inhibitor PPMP inhibits ORF2 protein polymerization and HEV assembly. First, the expression levels of HEV inside and outside HuH7-HEV-p6 cells were detected using isodensity gradient centrifugation with iodixanol, RT-qPCR, and protein immunoblotting. The results showed that in the extracellular viral cells, most eHEV particles in the control group cell culture supernatant were located in fractions 7-10, with a peak density of 1.10 g / cm³. 3 PPMP treatment significantly reduced the expression of HEV RNA and ORF2 protein in all eHEV fractions. Figure 3 A, Figure 3 B). Two populations were identified in the intracellular virions: the majority of nHEVs were located in fractions 14–16, with a peak density of 1.25 g / cm³. 3A small amount of eHEV was located in fractions 8-11. Gradient mapping of intracellular HEV RNA and ORF2 protein showed that PPMP effectively disrupted nHEV assembly. This primary assembly defect led to reduced intracellular eHEV formation and extracellular eHEV release. Figure 3 C Figure 3 D). Secondly, the multimerization status of ORF2 protein was detected using non-denaturing polyacrylamide gel electrophoresis. The results showed that PPMP and its analogue PDMP both eliminated intracellular ORF2 dimerization and significantly reduced the expression of extracellular ORF2 dimers and multimers. Figure 3 E, Figure 3 F). These data indicate that sphingolipids are involved in the dimerization and polymerization of the ORF2 protein, and the absence of sphingolipids inhibits the formation of the viral capsid, thereby blocking the assembly and release of HEV.

[0045] In one embodiment, the present invention demonstrates the co-localization binding of sphingolipids and ORF2 protein in the host cell Golgi apparatus. First, a bifunctional, photoreactive, clickable ceramide probe is used, comprising a diazacyclic propylene group for UV-induced crosslinking and an alkynyl group at the end of an alkyl side chain. The alkynyl group is used for a copper-catalyzed azido-alkynyl cycloaddition reaction to link azide biotin and for use in an immunoprecipitation assay with the target protein. Figure 4 A). Due to the strong interaction between ceramide and actin, we used actin as a positive control. The results showed that both actin and ceramide co-localized with ORF2 protein, and ceramide only interacted with ORF2 protein, while showing no significant interaction with ORF1 or ORF3 protein. Figure 4 B Figure 4 C Figure 4 D). PPMP treatment significantly reduced the interaction between ceramide and ORF2 protein ( Figure 4 E, Figure 4 F).

[0046] Next, immunofluorescence staining was used to further investigate the interaction between ceramide and ORF2 protein. The results showed that ORF2 protein and sphingosine co-localize (…). Figure 5 A). Control experiments were conducted using a no-UV irradiation group, a probe-free group, or a replication-deficient HEV-p6-GAD group to verify the specificity of the fluorescence signal. Furthermore, sphingosine and ORF2 protein exhibited strong co-localization in HuH7-HEV-p6 cells. Figure 5 B). Since sphingolipids are widely distributed in the endoplasmic reticulum, Golgi apparatus, endosomes, and lysosomes, we performed further subcellular localization analysis. The results showed that ORF2 and sphingosine are specifically co-localized in the Golgi apparatus. Figure 5 C Figure 5 D、 Figure 5 E, Figure 5 F). PPMP treatment not only significantly reduced the co-localization of ORF2 and pac-sph, but also reduced Golgi-specific co-localization (F). Figure 5 G, Figure 5 H, Figure 5 I, Figure 5 J、 Figure 5 These results indicate that sphingolipids and HEV ORF2 protein co-localize in the host cell Golgi apparatus.

[0047] In one embodiment, the HuH7-HEV-p6 cell model was constructed as follows: Cells were cultured in DMEM medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. The HuH7 cells (preserved in a laboratory cell bank) were cultured in a 37°C incubator containing 5% CO2. A recombinant plasmid carrying the full-length HEV genome (Kernow-C1 p6 clone; GenBank accession number JQ679013) was constructed. The HEV recombinant plasmid was linearized using the MluI restriction endonuclease, purified, and recovered as a transcription template. Capped HEV genomic RNA was synthesized using an in vitro transcription kit. The HEV genomic RNA was transfected into HuH7 cells using electroporation to construct the HuH7-HEV-p6 cell model. In this cell model, the HEV genomic RNA could stably replicate for 6-8 generations.

[0048] In one embodiment, sphingolipid inhibitor treatment was performed as follows: In the HuH7-HEV-p6 cell model, cells were treated with 6.25 μM PPMP and 2.5 μM MPA for 72 h, and cell culture supernatant and cell samples were collected.

[0049] In one embodiment, a protein immunoblotting experiment was performed as follows: Culture supernatant and cell protein samples were collected separately. The cell culture supernatant was directly added to non-denaturing loading buffer for subsequent detection. Cells were added to RIPA lysis buffer containing a mixture of protease inhibitors, lysed at 4°C for 30 min, and centrifuged at 12000×g for 5 min to collect the supernatant. Protein samples were added to protein denaturing loading buffer, heated in a 95°C metal bath for 5 min, and used for subsequent detection. Protein samples were then subjected to 10% SDS-PAGE. After separation by PAGE gel electrophoresis, the membrane was electroporated to a 0.22 μm PVDF membrane and blocked at room temperature for 1 h. Rabbit anti-ORF2 (1:3000) and mouse anti-β were used. The PVDF membrane was incubated overnight at 4 °C with actin (1:5000) primary antibody. The membrane was then incubated with HRP-labeled secondary antibody (1:5000) at room temperature for 1 h. Protein band images were acquired using a chemiluminescent developing solution and imaging system.

[0050] In one embodiment, animal experiments were conducted as follows: 13-week-old, approximately 70 g-weight, unspecified CMU long-clawed gerbils (purchased from Beijing Spif Biotechnology Co., Ltd.) were selected. Each gerbil was individually housed in a cage with ample drinking water and standard feed. The gerbils were randomly divided into a control group and an experimental group, with 7 gerbils in each group. During the rearing period, the animals' mental state, feeding, and activity were observed daily. HEV-containing cell lysates were obtained from HuH7 subclone S10-3 cell lysates after three freeze-thaw cycles. The long-clawed gerbils were infected with HEV via intraperitoneal injection. Each gerbil was inoculated with 3 × 10⁻⁶ cells. 7 Cell lysates containing viral copies were counted as day 0 of HEV infection. On day 3 post-infection, experimental gerbils were administered PPMP by gavage at a dose of 20 mg / kg, while the control group was administered the drug solvent by gavage at the same dose. Administration continued for 21 days. Blood samples were collected on days 0, 7, 14, and 21.

[0051] In one embodiment, the ELISA experiment is performed as follows: an appropriate amount of blood sample is taken to separate serum, and the HEV ORF2 antigen level in the serum is detected using the Wantai HEV-Ag ELISAPlus kit according to the prescribed steps.

[0052] In one embodiment, the isodensity gradient centrifugation experiment of iodixanol was performed as follows: ① Preparation of hepatitis E virus sample: After culturing HuH7-HEV-p6 cells for 15 days, the supernatant was collected and centrifuged at 1000 g for 10 min at 4°C. The supernatant was then collected and centrifuged at 1000 g for 30 min at 4°C. This centrifugation was repeated once. The supernatant was transferred to an ultracentrifuge tube and centrifuged at 100,000 g for 2 h at 4°C. The precipitate was collected and resuspended in PBS. ② Iodixanol isodensity gradient centrifugation: OptiPrep stock solution was diluted with Hank's balanced salt solution to prepare working solutions of iodixanol with mass / volume fractions of 8%, 16%, 24%, 32%, and 40%, respectively. The gradient solutions were slowly stacked from the bottom to the top of the tube at 4°C, with 700 μL of each concentration solution added to form a discontinuous iodixanol density gradient. 600 µL of the prepared hepatitis E virus sample was added to the top layer of the gradient and centrifuged at 37,000 rpm for 18 h at 4°C. Sixteen fractions were collected sequentially from the top of the gradient to the bottom of the tube for detection by RT-qPCR and Western blotting.

[0053] In one embodiment, RT-qPCR experiments were performed as follows: intracellular viral RNA was extracted using TRIzol; extracellular viral RNA was isolated and extracted using the FlashPure Viral DNA / RNA Extraction Kit II. The extracted RNA was reverse transcribed to synthesize cDNA. Quantitative detection was performed using 2×SYBR Green qPCR Master Mix reagent and a real-time quantitative PCR instrument. Viral standard RNA was serially diluted 10-fold to prepare a concentration of 1×10⁻⁶. 3 -1×10 8 Standards were prepared in copies / μL for each batch of experiments, and a standard curve was generated simultaneously. The viral genome copy number in the samples was calculated based on the standard curve. Extracellular viral load was expressed as viral copies per milliliter of culture medium; the relative expression level of intracellular HEV RNA was measured using a 23T / μL standard. ΔΔCt The calculation was performed using the method described above. The primer sequences used include SEQ ID NO. 1-4.

[0054] In one embodiment, the immunoprecipitation experiment was performed as follows: HuH7 cells were cultured to a density of approximately 80%. Overexpression plasmids of ORF1, ORF2, and ORF3 were transfected using Jetprime reagent. 72 h after transfection, the medium was changed to serum-free medium containing 2 µM pac-sph and incubated for 3 h. Cells were washed three times with PBS and then UV cross-linked for 5 min. Cells were lysed using 1% Triton X-100 solution containing a protease inhibitor. Lysis was performed at 4°C for 1 h, followed by centrifugation at 16000×g for 8 min. 200 µL of supernatant was transferred to a 2 mL centrifuge tube, 800 µL of methanol was added, and after mixing, 400 µL of chloroform was added, and after mixing again, 600 µL of ddH2O was added. The tube was centrifuged at 4°C at 15000 rpm for 2 min, and the upper aqueous phase was collected. Add 300 µL of methanol to the organic phase, mix well, and centrifuge at 15000 rpm for 5 min at 4°C. Discard the supernatant and air-dry the centrifuge tube on ice to obtain protein precipitate. Add 150 µL of PBS solution containing 4% SDS and incubate at 1200 rpm for 1 h on a shaker at 37°C to dissolve the protein precipitate. Add 25 mM CuSO4, 2.5 mM TBTA, 25 mM L-ascorbic acid, 10 mM azido-PEG3-biotin, and 1% SDS to the dissolved sample, and bring the volume to 1 mL with PBS. Incubate at 800 rpm for 3 h on a shaker at 37°C. Add 500 µL of pre-chilled methanol and centrifuge at 16000×g for 10 min. Discard the supernatant, resuspend the precipitate in 1 mL of pre-chilled methanol, and centrifuge at 16000×g for 10 min at 37°C. Discard the supernatant, add 80 µL of PBS solution containing 4% SDS, and incubate at 1200 rpm for 1 h on a 37°C shaker. Add another 1520 µL of PBS and centrifuge at 10000×g for 3 min. Use a portion of the supernatant as input sample after protein denaturation. Add magnetic beads to the remaining supernatant and incubate at room temperature for 1 h. Collect the magnetic beads by centrifugation at 3000×g for 1 min, wash 10 times with 1% SDS, and obtain eluate sample after protein denaturation. The collected input and eluate samples are used for the above-mentioned Western blotting experiments.

[0055] In one embodiment, the Pac-sph immunofluorescence staining experiment was performed as follows: HuH7 cells were seeded on a glass slide, and 10 μM pac-sph was added. 0.5 mL of PBS was added to the surface of the slide to cover the cells, and the slide was irradiated with UV light on ice for 2.5 min. Cells were fixed with methanol at -20℃ for 10 min. ① Click reaction: Fixed cells were extracted for 1 min at room temperature with 0.5 mL of a mixture of chloroform:methanol:acetic acid in a volume ratio of 10:55:0.75, repeated 3 times. Subsequently, a click reaction was performed with fluorescein 488 (azidofluorescein 488). 55 μL of freshly prepared click mixture (PBS solution of 1 mM ascorbic acid, 100 μM tris(benzyltriazolylmethyl)amine, 1 mM copper sulfate, and 2 μM fluorescein 488 (azidofluorescein 488)) was added to a coverslip, incubated at room temperature for 1 h, and then mounted. ② Colocalization experiment: The fixed cells were diluted with PBS (containing 4% bovine serum albumin and 0.02% Triton) with the primary antibody, incubated overnight at 4°C, and then incubated with the corresponding secondary antibody at room temperature for 1 h.

[0056] In one embodiment, data processing was performed as follows: GraphPad Prism 9 software was used for statistical analysis, and all experimental data were expressed as mean ± standard deviation of at least three independent replicates. Student's t-test was used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. The statistical difference labeling criteria are as follows: p<0.05; p<0.01; p<0.001; ns indicates no statistically significant difference between groups.

[0057] S102: Select the spatial structure of the sphingolipid-ORF2 protein complex and determine the binding site of the sphingolipid-ORF2 protein complex as the binding site of the targeted drug; In one embodiment, the method for determining the binding site of the sphingolipid-ORF2 protein complex includes: Predicting the interaction domains between sphingolipids and ORF2 protein using bioinformatics methods; Construct n truncated expression mutants of ORF2, where n is a natural number greater than or equal to 1; Based on the n truncated expression mutants of ORF2, it was found that the S and P2 domains of the ORF2 protein are key regions for interaction with sphingolipids. Bioinformatics methods were used to predict that the spatial conformation formed by the interaction between the conformational C1 pocket region at the interface of the S and P2 domains of the ORF2 protein and sphingolipid is the binding site of the sphingolipid-ORF2 protein complex. Construct n mutants with the C1 pocket region mutation, where n is a natural number greater than or equal to 1; Based on the mutants with the n C1 pocket region mutations, the binding sites of the sphingolipid-ORF2 protein complex include amino acid residues of ORF2 protein T170A, N171A, V470A, V501A, and V503A.

[0058] In one embodiment, the present invention employs bioinformatics methods to predict the binding site of the sphingolipid-ORF2 protein complex. First, the HEV ORF2 dimer structure PDB file and the sphingosine molecular structure are uploaded to CB-DOCK2. CB-DOCK2 automatically retrieves pocket structures within the HEV ORF2 dimer structure and then executes an Auto Docking program for molecular docking. The amino acid sites interacting with sphingosine are identified at the dimer binding interface.

[0059] In one embodiment, the present invention demonstrates that the sphingolipid-ORF2 protein interaction domain includes the S and P2 domains of the ORF2 protein. Sphingolipids typically interact with proteins via linear binding motifs defined by the sequence [V / I / T / L]XX[V / I / T / L][V / I / T / L]XX[V / I / T / L][F / W / Y]. Figure 6 A). The motif probability algorithm was used to scan the ORF2 protein sequence to determine the binding motif between sphingolipids and the ORF2 protein. The results showed that only one potential linear binding site was found in the secretory ORF2 protein, while no binding site was found in the capsid ORF2 protein. Figure 6 B Figure 6 C). However, immunoprecipitation experiments involving pac-sph and ORF2 showed that both ORF2 isoforms interacted with spholipids (C). Figure 6 D). This indicates that the interaction between sphingolipids and the ORF2 protein is not mediated by this linear motif.

[0060] Next, CB-Dock2 was used to predict potential ORF2 protein 3D binding sites. The results showed that the S domain, P1 domain, or P2 domain of the ORF2 protein may interact with sphingolipids. Figure 7 A, Figure 7 B Figure 7 C Figure 7 D、 Figure 7 E, Figure 7 F). To verify this prediction, we constructed a series of truncated ORF2 proteins with missing S, P1, and P2 domains, respectively. Figure 7 G). The expression of ORF2 in three truncated proteins was detected using Western blotting. The results showed that the loss of the P1 domain led to the failure of ORF2 truncated protein expression. Figure 7H). Immunoprecipitation assays were used to detect the interaction between sphingolipids and truncated proteins. The results showed that the deletion of either the S or P2 domain alone affected the interaction between ORF2 and sphingolipids. Figure 7 I). This indicates that the maintenance of the interaction between sphingolipid and ORF2 protein requires the participation of both the S domain and the P2 domain.

[0061] In one embodiment, this invention demonstrates that sphingolipids bind to the ORF2 protein in a conformational C1 pocket at the S-P2 domain of the ORF2 dimer interface, with binding sites including amino acid residues T170A, N171A, V470A, V501A, and V503A. First, further analysis of the interaction between the ORF2 dimer and sphingolipids using CB-Dock2 identified four potential binding pockets for the ORF2 protein and sphingolipids: C1, C2, C3, and C4. Figure 8 A, Figure 8 B Figure 8 C Figure 8 D、 Figure 8 E, Figure 8 F), and only pocket C1 can bridge the two independent structural domains S and P2 (F). Figure 8 G). Secondly, the C1 pocket structure was modeled and analyzed using CB-Dock2. The results showed that residues T170 and N171 (S domain) and E488, V470, W472, V501, V503, and Q508 (P2 domain) may be involved in the interaction between the ORF2 protein and sphingosine. Specifically, T170 and N171 residues in the S domain provide structural support for the pocket, while the predicted residues in the P2 domain directly interact with the alkyl chain or hydrogen bond donor or acceptor of sphingosine. Figure 9 A).

[0062] Next, to verify these findings, we constructed six ORF2 mutants through site-directed mutagenesis: M1 (T170A, N171A), M2 (E488A), M3 (V470A), M4 (W472A), M5 (V501A, V503A), and M6 (Q508A). We then used an immunoprecipitation assay to detect the sphingosine-binding affinity of the ORF2 protein in each mutant. The results showed that, compared with the wild type, the sphingosine-binding affinity of the M2, M4, and M6 mutants was not significantly different, while the M1, M3, and M5 mutants all showed a 30-40% downregulation of sphingosine-binding ability. Figure 9 B Figure 9 C), the M1+M3+M5 combination mutant completely disrupts the interaction between the ORF2 protein and sphingosine. Figure 9D). These results indicate that T170 and N171 are crucial for maintaining the C1 pocket architecture, while the hydrophobic interactions between the sphingosine alkyl chain and the V470, V501, and V503 residues are used to maintain the stability of the binding. Figure 9 E, Figure 9 F, Figure 9 G). These data indicate that sphingolipids bind to the ORF2 protein in a conformational C1 pocket at the S-P2 domain of the ORF2 dimer interface, with binding sites including amino acid residues T170A, N171A, V470A, V501A, and V503A.

[0063] In one embodiment, this invention demonstrates that mutations in the M1+M3+M5 amino acid residues lead to defects in HEV assembly and release. First, RNA mutants of M1, M3, M5, and M1+M3+M5 were constructed and introduced into HEV-p6 infectious clones, which were then used to infect HuH7 cells. The expression of HEV RNA and ORF2 protein in each mutant clone was then detected. Figure 10 A). The results showed that, compared with the wild type, although there was no significant change in intracellular HEV RNA levels, the M1, M3, and M5 single mutant clones significantly decreased extracellular HEV RNA expression levels. Furthermore, while the intracellular ORF2 expression levels of these three single mutant clones did not change significantly, extracellular ORF2 expression was significantly inhibited. The M1+M3+M5 mutant clone resulted in a slight decrease in intracellular RNA and ORF2 levels and almost completely inhibited extracellular HEV RNA release and ORF2 secretion. Figure 10 B Figure 10 C Figure 10 D). The M1+M3+M5 mutant clone disrupted nHEV assembly and inhibited the formation of eHEV within the cell and its release outside the cell. Figure 10 E, Figure 10 F, Figure 10 G, Figure 10 H).

[0064] Next, we injected the HEV M1+M3+M5 mutant strain into the liver of gerbils and measured the HEV RNA expression level in fecal samples. The results showed that the M1+M3+M5 mutant strain completely eliminated viral shedding in feces, and the HEV RNA levels in the liver, kidneys, and small intestine decreased to below the detection limit. Figure 11 A, Figure 11 B) Compared with the wild type, the long-clawed gerbil injected with the mutant strain showed significantly reduced inflammatory infiltration in the liver tissue and significantly lower levels of ORF2 protein in the tissue. Figure 11 C Figure 11 D). These results indicate that the M1+M3+M5 mutation leads to defects in HEV assembly and release, thereby effectively blocking HEV infection and transmission.

[0065] In one embodiment, the ORF2 isoform expression plasmid was constructed as follows: the HEV ORF2 protein isoform expression plasmid was constructed based on the pLVX-HEV ORF2 overexpression plasmid. The primers corresponding to the secretory and capsid ORF2 isoforms are SEQ ID NO.5-8 and NO.9-12, respectively.

[0066] In one embodiment, the HEV ORF2 protein mutant expression plasmid was constructed using the following method: Based on the pLVX-HEVORF2 overexpression plasmid, the HEV ORF2 protein mutant expression plasmid was constructed, and deletion mutations were performed using the Q5® site-directed mutagenesis kit and corresponding primers. The SEQ IDs of the primers corresponding to the ORF2 mutants (M1: T170A, N171A; M2: E448A; M3: V470A; M4: W472A; M5: V501A, V503A; M6: Q508A) are M1: NO.13-16; M2: NO.17-20; M3: NO.21-24; M4: NO.25-28; M5: NO.29-32; M6: NO.33-36, respectively. The SEQ IDs of the primers corresponding to the expression plasmids △S ORF2, △P1 ORF2, and △P2 ORF2 are as follows: △S: NO.49-50; △P1: NO.51-52; △P2: NO.53-54.

[0067] In one embodiment, the HEV-P6 mutant plasmid was constructed as follows: Mutation was performed on the full-length HEV-p6-Kernow / C1 plasmid using the NEB Q5 site-directed mutagenesis kit and corresponding primers. The corresponding primers have the following SEQ IDs: M1: NO.37-40; M3: NO.41-44; M5: NO.45-48.

[0068] In one embodiment, an animal model was constructed using the following method: ① In vitro transcription of HEV mutants: The mutant plasmid and the P6 wild-type plasmid were linearized using the MluI restriction endonuclease, respectively. Using these as templates, RNA was transcribed and capped under the drive of the T7 promoter. The capped RNA transcripts were purified by lithium precipitation, dissolved in enzyme-free sterile water, and the concentration was measured and stored. ② Animal rearing: 13-week-old, approximately 70 g-weight, unspecified CMU long-clawed gerbils were selected. Each gerbil was housed individually in a cage with sufficient drinking water and standard feed. They were randomly divided into a control group and an experimental group, with 5 gerbils in each group. During the rearing period, the animals' mental state, feeding, and activity were observed daily. ③ HEV inoculation: After thawing, the HEV RNA transcripts were injected into the liver of long-clawed gerbils. Four different injection sites were selected, and 10 μL was injected into each site. Fecal samples were collected 3, 7, 10 and 14 days after injection to detect HEV RNA expression levels. Liver, kidney and intestinal samples were collected 14 days after injection to detect HEV RNA expression levels. Histopathological staining and immunohistochemical staining were performed on gerbil liver samples.

[0069] In one embodiment, pathological histological staining and immunohistochemical staining experiments were performed as follows: Organ tissues were fixed in 10% neutral buffered formaldehyde solution for 3 days, embedded in paraffin, and cut into 5 μm thick sections for hematoxylin-eosin (HE) staining. Glutaraldehyde-fixed tissue sections were then subjected to immunohistochemical staining using ORF2 antibody. The staining results were observed under a microscope.

[0070] S103: Using a computer-aided drug screening method to obtain candidate drugs targeting the binding site; In one embodiment, the computer-aided drug screening method includes: Obtain the binding site of the sphingolipid-ORF2 protein complex; Based on the spatial structure of the binding site of the sphingolipid-ORF2 protein complex, compounds targeting the binding site of the sphingolipid-ORF2 protein complex were screened from the compound library. The selected compounds are molecularly docked with the ORF2 protein or sphingolipid to calculate the affinity or binding energy of the compounds to the ORF2 protein or sphingolipid, and a score is obtained for each compound. Candidate drugs are obtained by sorting the compounds according to the scores.

[0071] Optionally, the compound includes siRNA, shRNA, dsRNA, microRNA, antisense nucleic acid, antibody, polypeptide, protein analog, peptide analog or inorganic compound.

[0072] In one embodiment, the HEV ORF2 dimer structure PDB file is uploaded to CB-DOCK2, followed by the sequential upload of the molecular structures of the screened compounds. The validated pocket structures within the HEV ORF2 dimer structure are specified, and then the Auto Docking program is executed for molecular docking to observe whether the compounds interact with key amino acid sites.

[0073] In one embodiment, compounds from a compound library are docked with the binding sites of the sphingolipid-ORF2 protein complex. During docking, a computer simulates the interaction between the compounds and the binding sites and evaluates their binding affinity. Based on the docking results, compounds with strong target binding affinity can be screened as potential drug candidates. The determination of binding sites in virtual drug screening is crucial for the success of drug design. Therefore, multiple factors need to be considered when determining binding sites, such as the structural characteristics of the target and the binding modes of known ligands. Simultaneously, the binding sites need to be appropriately processed and optimized during virtual screening to improve the accuracy and efficiency of the screening.

[0074] In one embodiment, molecular docking is a method for drug design based on the characteristics of the receptor and the interaction between the receptor and the drug molecule. It primarily studies intermolecular interactions (such as ligand-receptor interactions) and predicts their binding modes and affinities. Molecular docking methods mainly focus on spatial matching and energy matching. Spatial matching refers to the geometric complementarity between the drug molecule and the receptor protein, while energy matching refers to the minimization of the interaction between the drug molecule and the receptor protein. Geometric matching calculations typically employ methods such as grid-based computation and fragment growth, while energy calculations utilize methods such as simulated annealing and genetic algorithms.

[0075] Based on the degree and method of simplification, molecular docking methods can be divided into rigid docking, semi-flexible docking, and flexible docking. In rigid docking, the conformation of the docking molecules remains unchanged during the calculation; only their spatial position and orientation are altered. Semi-flexible docking allows for partial conformational changes during the calculation. Flexible docking allows for even more conformational changes.

[0076] In molecular docking, affinity refers to the tightness of the binding between a molecule and its target. High affinity indicates a more stable binding, while low affinity indicates a less stable binding. Affinity is usually calculated, for example, by calculating the binding free energy (ΔG) or the binding constant (Kd). During molecular docking, affinity depends on the interactions between the molecule and the target, including hydrogen bonds, van der Waals forces, and electrostatic interactions. These interactions collectively determine the binding mode between the molecule and the target, thus affecting affinity. To assess affinity, scoring systems or methods are typically used to quantify the interaction between the molecule and the receptor. These scoring methods are based on different algorithms and physical models and can reflect the binding energy, interaction type, and affinity between the molecule and the receptor.

[0077] In one embodiment, the mode of screening drugs using computer-aided drug screening technology includes one or more of the following: proteins Small molecule docking, protein Protein docking, protein Nucleic acid testing docking.

[0078] In one embodiment, the protein Small molecule docking refers to the computational simulation process of docking the structures of proteins and small molecules together using specific algorithms and programs. This process can be used to study the interactions between proteins and small molecules, as well as their potential biological functions.

[0079] In protein In small molecule docking, software such as DOCK is commonly used for computational simulation. DOCK is a highly automated drug design software capable of docking small molecule ligands with biomolecular receptors. It employs a fragment-based scoring method, enabling fast and accurate docking. The basic algorithm of DOCK consists of two stages. The first stage is a low-precision stage, primarily searching for a coarse match between the small molecule ligand and the biomolecular receptor. The second stage is a high-precision stage, which considers all side-chain conformations and calculates more precise interaction energies. In the low-precision stage, DOCK randomly translates and rotates the small molecule ligand and performs a certain number of rigid body movements before calculating the interaction energy. After outputting the lowest conformation, it proceeds to the high-precision stage. In the high-precision stage, the program performs further optimization and adjustments to achieve more accurate docking.

[0080] In one embodiment, the protein Protein docking is a computational simulation process that uses specific algorithms and programs to dock the structures of two proteins together. This process can be used to study protein-protein interactions and their potential biological functions.

[0081] In protein RosettaDock is a commonly used docking software for protein docking. It employs a fragment-based scoring method to achieve fast and accurate protein docking. This software can precisely adjust the side-chain conformation during docking and considers various complex interactions, such as hydrogen bonds, ionic bonds, and hydrophobic interactions. RosettaDock's basic algorithm consists of two stages. The first stage is a low-precision stage, which mainly searches for the degree of skeletal shape compatibility between the two proteins. The second stage is a high-precision stage, which considers all side-chain conformations and calculates more precise interaction energies. In the low-precision stage, the program randomly translates and rotates a component of a protein molecule and performs a certain number of rigid body movements, then calculates the interaction energy. After outputting the lowest conformation, it enters the high-precision stage. In the high-precision stage, the program performs 50 MCMPCCcycles to rearrange the conformations and minimize the interaction energy, using this as the initial starting conformation.

[0082] In one embodiment, the protein Nucleic acid docking refers to the computational simulation process of docking the structures of proteins and nucleic acids (such as DNA or RNA) together using specific algorithms and programs. This process can be used to study the interactions between proteins and nucleic acids, as well as their potential biological functions.

[0083] In protein In nucleic acid docking, software such as NAflex is commonly used for computational simulation. NAflex is a software specifically developed for nucleic acid structure prediction and design, capable of accurate modeling and docking of DNA or RNA molecules. NAflex employs a fragment-based scoring method, enabling fast and accurate docking. It considers various complex interactions, such as hydrogen bonds, ionic bonds, and hydrophobic interactions, and can precisely adjust the side-chain conformation during docking. The basic algorithm of NAflex consists of two stages. The first stage is a low-precision stage, which mainly searches for a coarse match between the protein and nucleic acid. The second stage is a high-precision stage, which considers all side-chain conformations and calculates more precise interaction energies. In the low-precision stage, NAflex randomly translates and rotates the nucleic acid molecule and performs a certain number of rigid body movements before calculating the interaction energy. After outputting the lowest conformation, it enters the high-precision stage. In the high-precision stage, the program performs further optimization and adjustments to achieve more accurate docking.

[0084] In one embodiment, the molecular libraries used in virtual drug screening mainly include the following: DrugCLIP: Contains a large number of small molecules and proteins for protein-small molecule docking and genome-wide drug discovery.

[0085] ZINC: Contains over 250 million commercially available compounds for virtual screening of small molecules.

[0086] PubChem: Contains bioactive substances for virtual screening of small molecules.

[0087] DrugBank contains drugs and small molecules for drug design and discovery.

[0088] ChEMBL: Contains small molecules for drug discovery and chemical genomics research.

[0089] ChemDB contains a large number of known small molecules for chemical genomics research and drug discovery.

[0090] HMDB: Contains a large number of known small molecules used for chemical genomics research and drug discovery.

[0091] BindingDB contains a large number of known small molecules for chemical genomics research and drug discovery.

[0092] SMPDB contains a large number of known small molecules for chemical genomics research and drug discovery.

[0093] In addition, there are some commercial databases such as ChemDiv, Enamine, Lifechemicals, Specs, Chembridge, Maybridge, Microsource, Vitas Databases such as M and Interbioscreen are also frequently used for virtual drug screening.

[0094] In one embodiment, the drug screening method further includes: Obtain a system for expressing the ORF2 protein; The effectiveness of the compounds was verified by treating the system expressing the ORF2 protein with the compounds obtained from the screening.

[0095] Optionally, the system for expressing ORF2 protein includes a cell system expressing ORF2 protein, a subcellular system expressing ORF2 protein, a tissue system expressing ORF2 protein, a solution system expressing ORF2 protein, an organ system expressing ORF2 protein, or an animal system expressing ORF2 protein.

[0096] In one embodiment, the cell system expressing ORF2 protein, the subcellular system expressing ORF2 protein, the tissue system expressing ORF2 protein, the solution system expressing ORF2 protein, the organ system expressing ORF2 protein, or the animal system expressing ORF2 protein can be obtained through conventional purchasing channels known to those skilled in the art, or can be constructed through conventional construction methods known to those skilled in the art.

[0097] In one embodiment, the animal system expressing the ORF2 protein includes, but is not limited to, mice, rats, guinea pigs, rabbits, pigs, chickens, pigeons, monkeys, and dogs.

[0098] This invention also provides a computer-aided drug screening system based on the interaction between sphingolipids and the ORF2 protein. Specifically, the system includes: According to the acquisition unit, acquire sphingolipid and ORF2 protein data; The site determination unit selects the spatial structure of the sphingolipid-ORF2 protein complex and determines the binding site of the sphingolipid-ORF2 protein complex as the binding site of the targeted drug. The drug screening unit uses a computer-aided drug screening method to obtain candidate drugs that target the binding site.

[0099] Furthermore, the method for determining the binding site of the sphingolipid-ORF2 protein complex includes: Predicting the interaction domains between sphingolipids and ORF2 protein using bioinformatics methods; Construct n truncated expression mutants of ORF2, where n is a natural number greater than or equal to 1; Based on the n truncated expression mutants of ORF2, it was found that the S and P2 domains of the ORF2 protein are key regions for interaction with sphingolipids. Bioinformatics methods were used to predict that the spatial conformation formed by the interaction between the conformational C1 pocket region at the interface of the S and P2 domains of the ORF2 protein and sphingolipid is the binding site of the sphingolipid-ORF2 protein complex. Construct n mutants with the C1 pocket region mutation, where n is a natural number greater than or equal to 1; Based on the mutants with the n C1 pocket region mutations, the binding sites of the sphingolipid-ORF2 protein complex include amino acid residues of ORF2 protein T170A, N171A, V470A, V501A, and V503A.

[0100] Optionally, the method for obtaining the binding site of the sphingolipid-ORF2 protein complex is immunoprecipitation.

[0101] Optionally, the bioinformatics method includes molecular docking methods or prediction methods based on sequence and structural information.

[0102] Furthermore, the computer-aided drug screening method includes: Obtain the binding site of the sphingolipid-ORF2 protein complex; Based on the spatial structure of the binding site of the sphingolipid-ORF2 protein complex, compounds targeting the binding site of the sphingolipid-ORF2 protein complex were screened from the compound library. The selected compounds are molecularly docked with the ORF2 protein or sphingolipid to calculate the affinity or binding energy of the compounds to the ORF2 protein or sphingolipid, and a score is obtained for each compound. Candidate drugs are obtained by sorting the compounds according to the scores.

[0103] Optionally, the compound includes siRNA, shRNA, dsRNA, microRNA, antisense nucleic acid, antibody, polypeptide, protein analog, peptide analog or inorganic compound.

[0104] Furthermore, the method also includes an effectiveness verification unit: obtaining a system expressing the ORF2 protein; and treating the system expressing the ORF2 protein with the screened compound to verify the effectiveness of the compound.

[0105] Optionally, the system for expressing ORF2 protein includes a cell system expressing ORF2 protein, a subcellular system expressing ORF2 protein, a tissue system expressing ORF2 protein, a solution system expressing ORF2 protein, an organ system expressing ORF2 protein, or an animal system expressing ORF2 protein.

[0106] This invention also provides a computer-aided drug screening device based on the interaction between sphingolipids and ORF2 protein. Specifically, the device includes: The invention includes a memory and a processor, wherein the memory is used to store program instructions; and the processor is used to invoke the program instructions, which, when executed, implement the computer-aided drug screening method based on the interaction between sphingolipids and ORF2 protein as described above.

[0107] This invention also provides a computer-readable storage medium storing a computer program that, when executed by a processor, implements the computer-aided drug screening method based on the interaction between sphingolipids and ORF2 protein as described above.

[0108] This invention also provides a computer program product, which includes a computer program that, when executed by a processor, implements the computer-aided drug screening method based on the interaction between sphingolipid and ORF2 protein as described above.

[0109] The verification results of this verification embodiment show that assigning inherent weights to indications can moderately improve the performance of this method compared to the default settings.

[0110] Those skilled in the art will understand that, for the sake of convenience and brevity, the specific working processes of the systems, devices, and units described above can be referred to the corresponding processes in the foregoing method embodiments, and will not be repeated here.

[0111] In the several embodiments provided in this application, it should be understood that the disclosed systems, apparatuses, and methods can be implemented in other ways. For example, the apparatus embodiments described above are merely illustrative; for instance, the division of units is only a logical functional division, and in actual implementation, there may be other division methods. For example, multiple units or components may be combined or integrated into another system, or some features may be ignored or not executed. Furthermore, the coupling or direct coupling or communication connection shown or discussed may be through some interfaces; the indirect coupling or communication connection between apparatuses or units may be electrical, mechanical, or other forms.

[0112] The units described as separate components may or may not be physically separate. The components shown as units may or may not be physical units; that is, they may be located in one place or distributed across multiple network units. Some or all of the units can be selected to achieve the purpose of this embodiment according to actual needs.

[0113] Furthermore, the functional units in the various embodiments of the present invention can be integrated into one processing unit, or each unit can exist physically separately, or two or more units can be integrated into one unit. The integrated unit can be implemented in hardware or as a software functional unit.

[0114] Those skilled in the art will understand that all or part of the steps in the various methods of the above embodiments can be implemented by a program instructing related hardware. The program can be stored in a computer-readable storage medium, which may include: read-only memory (ROM), random access memory (RAM), magnetic disk or optical disk, etc.

[0115] Those skilled in the art will understand that all or part of the steps in the methods of the above embodiments can be implemented by a program instructing related hardware. The program can be stored in a computer-readable storage medium, such as a read-only memory, a disk, or an optical disk.

[0116] The computer device provided by the present invention has been described in detail above. For those skilled in the art, there will be changes in the specific implementation and application scope based on the ideas of the embodiments of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A computer-aided drug screening method based on the interaction between sphingolipids and ORF2 protein, characterized in that, The method includes: Obtain data on sphingolipids and ORF2 proteins; The spatial structure of the sphingolipid-ORF2 protein complex was selected, and the binding site of the sphingolipid-ORF2 protein complex was determined as the binding site of the targeted drug. Candidate drugs targeting the binding site were obtained using a computer-aided drug screening method.

2. The drug screening method according to claim 1, characterized in that, The method for determining the binding site of the sphingolipid-ORF2 protein complex includes: Predicting the interaction domains between sphingolipids and ORF2 protein using bioinformatics methods; Construct n truncated expression mutants of ORF2, where n is a natural number greater than or equal to 1; Based on the n truncated expression mutants of ORF2, it was found that the S and P2 domains of the ORF2 protein are key regions for interaction with sphingolipids. Bioinformatics methods were used to predict that the spatial conformation formed by the interaction between the conformational C1 pocket region at the interface of the S and P2 domains of the ORF2 protein and sphingolipid is the binding site of the sphingolipid-ORF2 protein complex. Construct n mutants with the C1 pocket region mutation, where n is a natural number greater than or equal to 1; Based on the aforementioned n C1 pocket region mutations, the binding sites of the sphingolipid-ORF2 protein complex include amino acid residues of ORF2 protein T170A, N171A, V470A, V501A, and V503A. Preferably, the method for obtaining the binding site of the sphingolipid-ORF2 protein complex is immunoprecipitation; Preferably, the bioinformatics method includes molecular docking methods or prediction methods based on sequence and structural information.

3. The drug screening method according to claim 1, characterized in that, The computer-aided drug screening method includes: Obtain the binding site of the sphingolipid-ORF2 protein complex; Based on the spatial structure of the binding site of the sphingolipid-ORF2 protein complex, compounds targeting the binding site of the sphingolipid-ORF2 protein complex were screened from the compound library. The selected compounds are molecularly docked with ORF2 protein or sphingolipid to calculate the affinity or binding energy of the compounds to target ORF2 protein or sphingolipid, and the scores of each compound are obtained. Candidate drugs are obtained by sorting the compounds according to the scores. Preferably, the compound includes siRNA, shRNA, dsRNA, microRNA, antisense nucleic acid, antibody, polypeptide, protein analog, peptide analog or inorganic compound.

4. The drug screening method according to claim 1, characterized in that, The drug screening method also includes: Obtain a system for expressing the ORF2 protein; The effectiveness of the compounds was verified by treating the system expressing the ORF2 protein with the compounds obtained from the screening. Preferably, the system for expressing ORF2 protein includes a cell system expressing ORF2 protein, a subcellular system expressing ORF2 protein, a tissue system expressing ORF2 protein, a solution system expressing ORF2 protein, an organ system expressing ORF2 protein, or an animal system expressing ORF2 protein.

5. A computer-aided drug screening system based on the interaction between sphingolipids and ORF2 protein, characterized in that, The system includes: The data acquisition unit acquires data on sphingolipids and ORF2 proteins. The site determination unit selects the spatial structure of the sphingolipid-ORF2 protein complex and determines the binding site of the sphingolipid-ORF2 protein complex as the binding site of the targeted drug. The drug screening unit uses a computer-aided drug screening method to obtain candidate drugs that target the binding site; Preferably, the method for determining the binding site of the sphingolipid-ORF2 protein complex includes: Predicting the interaction domains between sphingolipids and ORF2 protein using bioinformatics methods; Construct n truncated expression mutants of ORF2, where n is a natural number greater than or equal to 1; Based on the n truncated expression mutants of ORF2, it was found that the S and P2 domains of the ORF2 protein are key regions for interaction with sphingolipids. Bioinformatics methods were used to predict that the spatial conformation formed by the interaction between the conformational C1 pocket region at the interface of the S and P2 domains of the ORF2 protein and sphingolipid is the binding site of the sphingolipid-ORF2 protein complex. Construct n mutants with the C1 pocket region mutation, where n is a natural number greater than or equal to 1; Based on the aforementioned n C1 pocket region mutations, the binding sites of the sphingolipid-ORF2 protein complex include amino acid residues of ORF2 protein T170A, N171A, V470A, V501A, and V503A. Preferably, the method for obtaining the binding site of the sphingolipid-ORF2 protein complex is immunoprecipitation; Preferably, the bioinformatics method includes molecular docking methods or prediction methods based on sequence and structural information; Preferably, the computer-aided drug screening method includes: Obtain the binding site of the sphingolipid-ORF2 protein complex; Based on the spatial structure of the binding site of the sphingolipid-ORF2 protein complex, compounds targeting the binding site of the sphingolipid-ORF2 protein complex were screened from the compound library. The selected compounds are molecularly docked with ORF2 protein or sphingolipid to calculate the affinity or binding energy of the compounds to the ORF2 protein or sphingolipid, and scores are obtained for each compound. Candidate drugs are obtained by sorting the compounds according to the scores. Preferably, the compound includes siRNA, shRNA, dsRNA, microRNA, antisense nucleic acid, antibody, polypeptide, protein analog, peptide analog or inorganic compound; Preferably, the method further includes an effectiveness verification unit: obtaining a system expressing the ORF2 protein; and treating the system expressing the ORF2 protein with the screened compound to verify the effectiveness of the compound. Preferably, the system for expressing ORF2 protein includes a cell system expressing ORF2 protein, a subcellular system expressing ORF2 protein, a tissue system expressing ORF2 protein, a solution system expressing ORF2 protein, an organ system expressing ORF2 protein, or an animal system expressing ORF2 protein.

6. A computer-aided drug screening device based on the interaction between sphingolipids and ORF2 protein, characterized in that, The device includes: A memory and a processor, wherein the memory is used to store program instructions; and the processor is used to invoke the program instructions, which, when executed, implement claim 1. The drug screening method described in any one of the following 4.

7. A computer-readable storage medium, characterized in that, The computer-readable storage medium stores a computer program, which, when executed by a processor, implements claim 1. The drug screening method described in any one of the following 4.

8. A computer program product, characterized in that, The computer program product includes a computer program that, when executed by a processor, implements the drug screening method according to any one of claims 1-4.

9. Any one of the following products: 1) A sphingolipid-ORF2 protein complex, characterized in that, The sphingolipid-ORF2 protein complex is the sphingolipid-ORF2 protein complex described in claim 1; 2) A drug obtained by any one of the drug screening methods according to claims 1-4.

10. Any of the following applications: 1) The application of a protein complex, characterized in that, The complex is the sphingolipid and ORF2 protein complex as described in claim 9, and the application includes: the application of the complex in regulating hepatitis E virus replication and / or release; 2) The application of a drug, characterized in that the drug is the drug as described in claim 9, and the application includes: the application of the drug in the prevention or treatment of hepatitis E virus.

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